Methods and compositions for eliminating allergens and allergen-producing organisms

ABSTRACT

Methods are provided for making a treatment composition by loading active components into a high pressure vessel and pressurizing the high pressure vessel with carbon dioxide to reach a pressure within the high pressure vessel of about 400 pounds per square inch to about 1,070 pounds per square inch. The active components can include a protein denaturant and a surfactant, and optionally an acaricide. In one particular embodiment, this method can be used to clean a substrate, by loading the substrate into the high pressure vessel prior to pressurizing with carbon dioxide. Methods are also provided for treating a substrate to clean it from dust mites by delivering dry ice particles to the substrate, and vacuuming the substrate. Treatment compositions are also generally provided.

PRIORITY INFORMATION

The present application claims priority to U.S. Provisional Patent No.61/340,897 filed on Mar. 24, 2010 titled “Methods and Compositions forEliminating Allergens and Allergen-Producing Organisms” of Matthews, etal. and U.S. Provisional Patent No. 61/398,812 filed on Jul. 1, 2010titled “Reducing Dust Mite Populations and Allergen Levels Using HighSpeed Dry Ice Jets” of Matthews, et al., the disclosures of which areincorporated by reference herein.

FIELD OF INVENTION

The technology relates to the prevention and removal of harmfulallergens from substrates in a home leading to improved air quality toinhibit allergic reactions and other health problems in humans.

BACKGROUND OF INVENTION

More than 50 million Americans suffer from allergies, making them thesixth leading cause of chronic disease in the United States, andresponsible for 3.8 million lost work or school days per year. Commonhouse dust is a leading cause of allergies. Although there are manycomponents in house dust that may be allergenic, the most common culpritby far is the dust mite, which is second only to pollen overall incausing allergic reactions. More than 15 groups of house dust miteallergens (proteins) have been identified from extracts and feces ofdust mites. The group 1 and group 2 proteins are the major allergens,accounting for 60 to 90% of the activity.

Furthermore, dust mite allergens are closely related to the onset ofasthma. Asthma is a serious and potentially fatal condition that isespecially critical in children under age 13. The American LungAssociation estimates that 32.5 million Americans (approx. 11.2%) havebeen diagnosed with asthma. 14.2% of American children between 5 and 17years of age had asthma is in 2005. It has been shown that most childrenwith exacerbation of asthma have been exposed to high levels of miteallergens, and that continuous exposure can result in hospitalreadmission. The highest levels of allergens are found in bedding(blankets, sheets, spreads, pillow cases, etc.), placing humans at highrisk of inhaling the offending proteins. A nationwide survey conductedby the National Institute of Environmental Health Sciences (NTRHS) andthe US Department of Housing and Urban Development in 1998 and 1999reports that 84% of U.S. homes have detectable levels of mite allergens;about half have levels sufficient to trigger allergic reactions; and aquarter of U.S. homes have high enough allergen level to cause asthma.The cost of asthma in 2007 is projected to reach $19.7 billion ($14.7direct, $5 indirect). In New York's Harlem neighborhood, 25.5% ofchildren under age 13 suffer from asthma. The sensitization level of thegroup 1 allergen in adults is 2 μg allergen/g dust, and 10 μg allergen/gdust enhances asthma symptoms. For children, the sensitization levelcould be as low as 50 ng/g, far below the general sensitization level.

Dust mites are eight-legged arachnids, relatives of the spider. TheAmerican house dust mite (Dermatophagoides farinae, Df) and the Europeanhouse dust mite (Dermatophagoides pteronyssinus, Dp) are the two mostcommon species in the United States. Dust mites progress through theegg, larval and nymph stages in about 25 days to become microscopicadults about 0.3 mm in length. Adults live two to three months, duringwhich time the female can lay 25 to 50 eggs every two to three weeks.They feed on shed scales of human skin. Only environments with arelative humidity of less than 50% year round are safe from theseallergy causers. Mites live in carpet, upholstered furniture, andbedding. The allergenic proteins are actually present in dust mite fecesand each dust mite produces about twenty waste particles per day. Morethan 100,000 particles can be present in a single gram of dust.Particles continue to cause allergy symptoms long after the dust mitesthemselves are dead. Mite allergens can cause or exacerbate three majordiseases: asthma, perennial rhinitis, and atopic dermatitis. Theconsequences of which can be fatal.

More than 15 groups of house dust mite allergens have been identifiedfrom extracts and feces of Dp and Df dust mites. The group 1 (Der p 1and Der f 1) and group 2 (Der p 2 and Der f 2) proteins are the majorallergens, accounting for 60 to 90% of IgE binding activity of the dustmite sera. The group 2 allergen levels are comparable to the levels ofthe group I allergens. The group 1 allergens are proteins (MW 25 kDa)with cysteine protease activity. Proteolytic activity is an importantfactor for sensitization by Der p 1. Der p 1 cleaves the CD23 IgEreceptors on B-cells and the CD25 subunit of the IL-2 receptor onT-cells, which further promote the Th2 response and IgE-mediatedhypersensitivity (allergy).

At present, there are few effective options for patients with severeallergic or asthmatic reactions. Allergen avoidance (i.e. relocation toallergy-free environments) has been shown to result in reduced asthmasymptoms. Obviously, moving people and families into specialenvironments is both expensive and socially disruptive. Conventionalallergen avoidance measures include washing of fabrics in hot water,using allergen impermeable covers, regular vacuuming, and elimination ofcarpet and upholstered furnishings. Washing is recommended to controldust mite allergen levels. Washing can remove soluble allergens, and hotwater will kill dust mites if the temperature is higher than 55° C. Manyitems of bedding cannot be washed in a household washer, and ordinaryU.S. household washers operate with warm or cold water that does notensure mite death. D. pteronyssinus is highly resistant to warm water,detergent and chlorine. A 4 hour soak in a 35° C. detergent and chlorine(0.35%) solution achieved only 34% mortality of D. pteronyssinus. Evenwith the addition of 0.03% benzyl benzoate, approximately 50 out of10,000 mites survived. Additional concerns about washing include thetransference of mites between infested and mite-free items, and the factthat some fabrics will shrink or be damaged when washed at 55° C.Chemical-based dry cleaning effectively kills mites and can be used toclean delicate clothing and some bedding items, but has been shown toreduce the allergen activity by only 70%, because the residual proteinsare not deactivated by dry cleaning. Semi-impermeable mattress coversare said to control allergen levels for at least 12 weeks, leading toimproved symptoms. However, this method is not low-maintenance as itrequires regular washing of the case at T>55° C. and periodic wiping.

Using acaricides to kill dust mites, therefore eliminating the source ofallergens, is another method that has been proposed to reduce allergenlevels. Heller-Hauput and Busvine tested 15 chemicals for ability tokill D. farinae. In the order of potency, Lindane, pirimiphosmethyl, andbenzyl benzoate are the most effective. Toxicity studies and a historyof use for treatment of scabies indicate that Lindane and benzylbenzoate can safely be used to kill dust mites. However, acaricides donot deactivate residual allergy-causing mite proteins. Solid benzylbenzoate foam and powders (60 g/m2, then vacuumed) have been tested onmattresses and blankets in Germany for their efficiency of reducing Derp 1 and der f 1 allergens. The tests revealed that powered benzylbenzoate only works on blankets possibly due to the difficulty of thesolid material penetrating the mattresses which are much thicker thanblankets. A similar study in Italy reported that benzyl benzoate in foamform is also not effective in decreasing dust mite allergen againpossibly due to insufficient penetration.

Prior art indicates that dense phase CO₂ can kill various types ofcells, including microorganisms (bacteria, bacterial spores, fungus) andmammalian cells, with the help from selected liquid chemicals includingwater and hydrogen peroxide. It takes hours to kill resistant speciessuch as bacterial spores. However, it only takes minutes to kill animalcells such as mammalian cells. Therefore, dense phase CO₂ can killliving dust mites quickly.

It is the protein secreted by dust mites that are the main cause ofallergic reactions and asthma. These proteins secreted byDermatophagoides pteronyssinus include a spectrum of mite allergensdenominated as der p1 to der p9. Among these, der p1 and der p2 are themajor allergens. Partial inactivation of proteins by dense phase CO₂ hasbeen widely documented in the literature. However, no completeinactivation of enzymes with CO₂ has been previously reported.

Current state of the art measures to prevent mite allergen-induceddiseases include frequent vacuuming with high filtration vacuumcleaners, UV light irradiation, topical application of an insecticide tokill the dust mite, covering all beds with micro-porous dust-proof orallergen impermeable covers, weekly washing of bedding with hot waterand freezing non-washable beddings overnight, and removing carpets fromall rooms. However most prior art suffers from many drawbacks. Vacuumcleaners and insecticides suffer from the inability to penetrate deepinto carpets. Also, carpets usually cannot be removed and frozen; or bewashed with hot water. It is not feasible to cover entire householdcarpets with impermeable covers. In addition, water washing, even withhot water, will not kill dust mites; and impermeable covers appearclinically ineffective in adults with asthma.

As such, a need exists for the eradication of dust mites that overcomesthe disadvantages of prior art methods.

SUMMARY

Objects and advantages of the invention will be set forth in part in thefollowing description, or may be obvious from the description, or may belearned through practice of the invention.

Methods are generally provided for making a treatment composition byloading active components into a high pressure vessel and pressurizingthe high pressure vessel with carbon dioxide to reach a pressure withinthe high pressure vessel of about 400 pounds per square inch to about1,070 pounds per square inch. The active components can include aprotein denaturant and a surfactant, and optionally an acaricide. In oneparticular embodiment, this method can be used to clean a substrate, byloading the substrate into the high pressure vessel prior topressurizing with carbon dioxide.

Treatment compositions are also generally provided that include (e.g.,comprise, consist essentially of, or consist of) a surfactant, a proteindenaturant, an optional acaricide, and a dense phase carbon dioxide,wherein the composition has a pressure of about 400 pounds per squareinch to about 1,070 pounds per square inch.

Methods are also generally provided for treating a substrate to clean itfrom dust mites by delivering dry ice particles (e.g., including frozencarbon dioxide and optionally an acaricide, a protein denaturant, and/ora surfactant) to the substrate, and vacuuming the substrate.

Other features and aspects of the present invention are discussed ingreater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

A full and enabling disclosure of the present invention, including thebest mode thereof to one skilled in the art, is set forth moreparticularly in the remainder of the specification, which includesreference to the accompanying figures, in which:

FIG. 1 shows an exemplary allergen denaturation apparatus;

FIG. 2 shows a chart of inactivation results of scCO₂ with differentadditives at 4000 psi, 60° C., 5 hrs according to the Examples of thepresent disclosure;

FIG. 3 shows a chart of temperature dependence of CO₂+LS-54+1% tannicacid treatment (4000 psi, 5 hrs) according to the Examples of thepresent disclosure;

FIG. 4 shows an exemplary allergen and endotoxin solubilization andremoval apparatus according to the Examples of the present disclosure;

FIG. 5 shows a chart of endotoxin removal with CO₂ (liquid andsupercritical) and mixtures of water and surfactant Ls-54; and

FIG. 6 shows a chart of endotoxin removal for disks treated with liquidCO₂+Ls-54 and water at 4,000 psi & 2 hr; and 2,000 psi & 4 hr in the 1 Lpressure vessel.

Repeat use of reference characters in the present specification anddrawings is intended to represent the same or analogous features orelements of the present invention.

DETAILED DESCRIPTION

Reference now will be made to the embodiments of the invention, one ormore examples of which are set forth below. Each example is provided byway of an explanation of the invention, not as a limitation of theinvention. In fact, it will be apparent to those skilled in the art thatvarious modifications and variations can be made in the inventionwithout departing from the scope or spirit of the invention. Forinstance, features illustrated or described as one embodiment can beused on another embodiment to yield still a further embodiment. Thus, itis intended that the present invention cover such modifications andvariations as come within the scope of the appended claims and theirequivalents. It is to be understood by one of ordinary skill in the artthat the present discussion is a description of exemplary embodimentsonly, and is not intended as limiting the broader aspects of the presentinvention, which broader aspects are embodied exemplary constructions.

In general, treatment compositions, along with the methods of making andusing, are generally disclosed for eradicating living dust mites anddenaturing allergenic proteins secreted by dust mites in substrates thatare known to harbor these allergenic sources. For example, in oneembodiment, the composition includes a combination of pressurized carbondioxide, a surfactant, and/or a protein denaturant. In particularembodiments, the compositions include pressurized carbon dioxide,surfactants, acarcides, and/or protein denaturants, such that thecompositions are configured to kill dust mites and/or denatureallergenic proteins and/or solubilize or remove allergenic proteins andendotoxins. Additionally, methods and compositions are generallydisclosed for solubilization and removal of dust mite allergens andbacterial endotoxin with compositions comprising the combination ofpressurized carbon dioxide and a surfactant.

The methods and treatment compositions presently disclosed generally usedense phase carbon dioxide (CO₂) as a solvent medium. This dense phaseCO₂ uniquely aids in the formation and delivery of microemulsions ofsurfactants and/or oxidizing molecules (e.g., hydrogen peroxide,chlorine dioxide, or mixtures thereof). The oxidizing molecules cansubsequently denature allergenic proteins and enhance the killing ofallergen producing organisms. In one particular embodiment, the use ofdense phase CO₂, with surfactants and mite allergen denaturant (e.g.,tannic acid), promotes the inactivation of allergenic proteins.

I. Composition

As stated, treatment compositions are disclosed that generally includepressurized carbon dioxide, a surfactant, an acaricide, and/or a proteindenaturant. Other allergy controlling agents (e.g., amines, hopsextracts, anti-allergenic iodo derivatives, quaternary ammoniumcompounds, glycol ether, glycol ether ester, or mixtures thereof) canalso be included in the composition to promote the inactivation ofallergenic proteins.

The term “dense phase carbon dioxide” can also be referred to as“high-pressure carbon dioxide,” “high-pressure CO₂,” “dense-phase carbondioxide,” and “dense-phase CO₂” refers to pressurized carbon dioxide,either in its liquid state near, but below, the critical temperature andnear the critical pressure or to supercritical carbon dioxide, aboveboth the critical temperature and critical pressure. For example, in oneaspect, the pressure can be from about 400 pounds per square inch (about27.6 bar) to about 4,000 pounds per square inch (about 275 bar). Inanother aspect, the pressure can be from about 500 pounds per squareinch (about 34.5 bar) to about 850 pounds per square inch (about 58.6bar). In a further aspect, the pressure can be from about 600 pounds persquare inch (about 41.36 bar) to about 750 pounds per square inch (about51.7 bar). Alternatively, the pressure can be about 2000 pounds persquare inch to about 4000 pounds per square inch. The temperature rangecan be about 0° C. to about 60° C., such as from about 10° C. to about25° C.

One or more surfactants can be included with the CO₂ solvent to enhancethe formation of micro emulsions. The surfactant(s) that can be includedwithin the compositions include, but are not limited to,fluorosurfactants, ionic surfactants, silicon-based surfactants (e.g.,siloxane surfactants), hydrocarbon surfactants, or mixtures thereof. Inparticular embodiments, the surfactant(s) can have a concentration rangeof about 0.2% by volume to about 10% by volume of the treatmentcomposition, such as about 0.5% by volume to about 5% by volume.

Acaricides are pesticides that kill members of the Acari group, whichincludes ticks and mites. Their inclusion in the composition enhancesthe elimination of allergy producing organisms (e.g., mites). Suitableacaricides can include, but are not limited to permethrin, benzylbenzoate, ivermectin, antibiotic miticides, carbamate miticides,formamidine miticides, organochlorine, organophosphate miticides,diatomaceous earth, dicofol, or mixtures thereof. Particularly suitableacaricides that can be included within the composition include, but arenot limited to, benzyl benzoate, permethrin, or mixtures thereof. Inparticular embodiments, the acaricide(s) can have a concentration rangeof about 0.01% by volume to about 5% by volume of the treatmentcomposition, such as about 0.05% by volume to about 3% by volume.

The protein denaturant of the compositions can be included as a miteallergen denaturant, targeting the proteins and other organic compoundsformed by mites. Such protein denaturants can include, but are notlimited to, ethanol, hydrogen peroxide, benzyl benzoate, and tannicacid. In addition, synthetic non-oxidizing protein inhibiters, such asPTL 11028, a peptide-based inhibitor, can be included in the compositionto promote the inactivation of allergenic proteins. In particularembodiments, the protein denaturant(s) can have a concentration range ofabout 0.001% by volume to about 3% by volume of the treatmentcomposition, such as about 0.01% by volume to about 1% by volume.

II. Methods of Making the Treatment Compositions

According to one particular embodiment, the treatment compositions canbe made by loading the desired active components (e.g., proteindenaturant(s), surfactant(s), acaricide(s), etc., and mixtures thereof)into a high pressure vessel and pressurizing the vessel with CO₂ gas.For example, the internal pressure within the vessel, upon adding CO₂gas can be about 400 pounds per square inch (about 27.6 bar) to about1,070 pounds per square inch (about 73.7 bar), as discussed above.

In one particular embodiment, the CO₂ gas, when loaded into the vessel,has a purity of about 90% by volume or more (e.g., about 95% to 100% byvolume). For instance, the CO₂ gas can be substantially pure CO₂ gas, asin substantially free from other molecules. As used herein, the term“substantially free” means no more than an insignificant trace amountpresent and encompasses completely free (e.g., 0% up to 0.01% byvolume).

The high pressure vessel is generally designed to withstand therelatively high pressures involved with the dense phase carbon dioxidecomposition, such as up to about 1500 pounds per square inch. In oneparticular embodiment, the vessel is large enough to accommodate thesubstrates themselves. For example, the vessel can be a modified washermachine-like vessel configured to withstand relatively high pressures.

An exemplary allergen denaturation apparatus is depicted in FIG. 1 asused in the Examples. Gas for preparation of a high-pressure orsupercritical fluid is supplied to the high pressure vessel 6 from gastank 1 via gas line 13. Supercritical CO₂ refers to pressurized, fluidcarbon dioxide at or above the critical temperature (about 31.1° C.) andat or above the critical pressure (about 73.8 bar), while supercriticalfluid refers to a pressurized, fluid gas at or above the criticaltemperature and at or above the critical pressure. High-pressure fluidand dense-phase fluid refer to pressurized, liquid gas near but belowthe critical temperature and near but below the critical pressure. Inone aspect, the pressure can range from 35% to 99% of the criticalpressure of the gas (e.g. from 40% to 85% of the critical pressure, from60% to 75% of the critical pressure.) Gas line 13 is an air-tight hollowtube constructed so as to withstand the required pressures forhigh-pressure and supercritical fluids. Pump inlet valve 2 is positionedin fluid communication with gas line 13 and positioned intermediate gastank 1 and syringe pump 3. Accordingly, pump inlet valve 2 can controlthe flow of gas from gas tank 1.

Syringe pump 3 functions to provide gas from gas tank 1 to high pressurevessel 6 as well as to provide the pressures for high-pressure orsupercritical fluids. In one aspect, syringe pump 3 is calibrated toprovide a selected pressure. Inlet valve 5 can control the flow of gasfrom syringe pump 3 and is located along gas line 13 between syringepump 3 and high pressure vessel 6.

The high pressure vessel 6 is placed inside an environmental chamber 4.Environmental chamber 4 is programmed to raise or lower the temperaturewith desired fashion or to keep constant temperature. High pressurevessel 6 has one hand-tight cap 7 and one stainless steel filter 8 ateach end of the vessel body. Liquid additives 9 are directly placed onthe lower filter 8. When CO₂ is supplied through the gas line 13 andpasses through the lower filter 8, CO₂ bubbles through the liquidadditives 9 and solubilize the additives into CO₂ phase. The pressureinside the high pressure vessel is measured with a pressure gauge 11connected to the top cap 7. A rupture disc 12 is also connected to thetop cap 7 to prevent over pressurizing the vessel 6. Vent valve 14 isconnected to the bottom cap 7 of the vessel and is used to release CO₂pressure and vent liquid additives 9 from the vessel 6 at the end of anexperiment.

Proteins or allergens are contained in a glass vial 10. The glass vial10 is attached to the top filter with a string and suspended in the CO₂phase. CO₂ can freely enter and leave the vial through the opening ontop of the vial.

III. Methods of “Washing” Substrates in the Treatment Composition

Methods are also generally disclosed for using the disclosedcompositions for denaturing, solubilizing, and removing allergenicproteins. Specifically, the compositions can be used to help eradicatesubstrates from mites and their by-products. The substrates applicableto this method include, but are not limited to, beddings (e.g., pillows,comforters, mattresses, pads, covers), carpets, soft furnishings (e.g.,area rugs, curtains, drapes), and/or furniture cushions (e.g., pillows,throws, seat covers, furniture covers).

In one particular embodiment, the substrate(s) can be loaded into a highpressure vessel, which is then filled with the treatment composition.For example, the protein denaturant and/or surfactant and/or acaricidesand/or other components can be loaded into the high pressure vesselafter or with the substrate(s), and CO₂ gas can be used to pressurizethe gas. The vessel can be held at the desired conditions (temperature,pressure, agitation) as discussed above, prior to finally releasing thepressure. For instance, in a particularly suitable “washing cycle”, thesubstrate can be exposed to the treatment composition for a period aboutan hour or less, such as about 20 minutes to about 45 minutes, with thetreatment pressure being about 500 pounds per square inch to about 1000pounds per square inch at a temperature of about 5° C. to about 30° C.Finally, the substrate(s) can be rinsed with high pressure CO₂ gas toremove residual components (e.g., protein denaturant and/or surfactantand/or acaricides, etc.).

IV. Methods Using Dry Ice Pellets

Alternatively, dry ice pellets (i.e., frozen carbon dioxide) can be usedto loosen and/or kill dust mites and their feces in a substrate (e.g., acarpet). For example, solid dry ice particles (e.g., created accordingto the method described in U.S. Pat. No. 7,293,570 of Jackson, which isincorporated by reference herein) can be ejected from nozzles. Forexample, the nozzles can generate sheer stress up to 55 MPa. These soliddry ice particles impact with dust mites and dust mite feces attached tocarpet fibers and dislodge them from the carpets. Next, a high efficientvacuum system can remove the dislodged dust mites and mite feces whichcontain dust mite allergens. In addition, by impacting dust mites withultra low temperature dry ice particles (e.g., −109° F.), the dust miteswill be instantaneously frozen and killed. At room temperature, dry iceparticles sublime quickly to gaseous carbon dioxide, leaving no solventresiduals.

The dry ice particles can, in one embodiment, have a size (i.e., anaverage diameter) of 1 micron to about 500 microns, such as about 10microns to about 200 microns.

In one particular embodiment, an acaricide, surfactant, and/or proteindenaturant (such as described above) can be pre-dissolved in liquid CO₂upstream of the nozzle. For example, upon formation of dry ice particlesincluding an acaricide, the acaricide is frozen into the particles andis forced deep into the carpet by the velocity of the stream. The CO₂then sublimes, leaving the less-volatile acaricide safely and deeplyimbedded into the carpet. The acaricide serves to prevent re-infestationof dust mites, thus providing even longer-term protection.

As such, this method allows deeper and more thorough cleaning of carpetsthan conventional vacuuming or UV irradiation. In addition, the methodpresented in this invention will greatly reduce the dust mite populationand allergen levels in carpets. Treatment of such articles with theprocess disclosed herein will reduce the cost of treatment and alleviatesuffering in adults, children, and infants who are susceptible toallergies.

Thus, a method of treatment can include using solid carbon dioxideparticles to loosen dust mites from carpets so that the dust mites canbe removed with vacuum and/or using solid carbon dioxide particles tokill dust mites by lowering their temperature to about −78.5° C. orlower. Additionally, the methods can include using solid carbon dioxideparticles to deliver an acaricide into the carpet, allowing residualacaricide to inhibit re-infestation of dust mites.

The components used to prepare the disclosed compositions as well as thecompositions themselves used within the methods are disclosed herein. Itis understood that when combinations, subsets, interactions, groups,etc. of these materials are disclosed, the various individual andcollective combinations and pe mutation of these compounds may arespecifically contemplated even if they are not explicitly describedherein. For example, if a particular compound is disclosed and discussedand a number of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the disclosed compositions. Thus, if there are a variety ofadditional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures which can perform the same function which arerelated to the disclosed structures, and that these structures canultimately achieve the same result.

EXAMPLES

The following examples are meant to illustrate the invention describedherein and are not intended to limit the scope of this invention.Current laboratory work on inactivating enzymes has shown that 80-90% ofenzymatic activity of trypsin, a model protease, can be reduced withsupercritical CO₂ containing a microemulsion of selected oxidizing(H₂O₂) and non-oxidizing (1% tannic acid) agents.

Example of Protein Denaturation

Proteins/Allergens

In this example, trypsin was used as a model protein to demonstrate thecapability of the disclosed allergen denaturation method. The trypsinwas extracted from bovine pancreas and supplied as salt free lyophilizedpowder.

Quantifying Denaturation

The enzymatic activity of trypsin was quantified with a BAEE(N-a-Benzoyl-L-Arginine Ethyl Ester) assay: The amount of trypsin wasmeasured by the rate of increase in the optical density of BAEE solutionas a result of enzymatic degradation of BAEE. Four reagents wereprepared for the BAEE assay. Reagent A was 67 mM sodium phosphate bufferat pH 7.6 at 25° C. Reagent B was 25 mM BAEE in Reagent A. Reagent C was1 mM hydrochloride acid. And Reagent D was freshly made trypsin samplein cold Reagent C.

Three milliliters of Reagent B and 0 2 mL of reagent D were quicklymixed inside a cuvette and measured with a IN-V is spectrophotometer.The increase in the optical density at 253 nm was measured forapproximately 5 min and compared with a standard sample to determine theenzymatic activity of the trypsin sample.

Procedure

Prior to the denaturation experiment, milligram levels of trypsin powderwere transferred into a clean 2 mL glass vial. The septum of the vialcap had been replaced with a nylon filter to prevent escapes of trypsinparticles, and to allow the free flow of CO₂ into and out of the vial.Three milliliters of a surfactant LS-54 and 2 mL of selected proteindenaturant (e.g. ethanol, 1% tannic acid, 30% hydrogen peroxide) wereplaced directly on top of the lower filter. The surface tension of thisliquid mixture prevented it from flowing downwards through the filter.The trypsin containing vial was attached to the top filter with a cottonstring to prevent direct contact between liquid denaturant and protein,so as to avoid false positive results.

At the beginning of the experiment, the environmental chamber waspreheated to desired temperature (40° C. and 60° C. in this example, butother temperatures can be used) and the syringe pump raised the CO₂pressure to a pre-set value (e.g. 4000 psi). Then the vent valve wasclosed and inlet valve was slowly opened to allow CO₂ to flow throughthe filter and the liquid mixture, where the liquid mixture dissolvedinto the CO₂ phase and create water-in-CO₂ microemulsions.

The temperature and pressure were maintained for the predeterminedamount of time. Then the inlet valve was closed. The pressure chamberwas slowly depressurized through the vent valve untile the vesselreached atmospheric pressure.

The trypsin containing vial was immediately removed from the vessel.Trypsin was dissolved in cold 1 mM hydrochloric acid solution andassayed with the BAEE procedure.

Results and Discussion

Different additives and combinations among them (water, 30% H₂O₂, 1%tannic acid, LS-54, LS-54+H₂O, LS-54+1% tannic acid, LS-54+30% H₂O₂)were examined for their efficacy of denaturing trypsin at 4000 psi, 60°C. for 5 hours. Among these additives, water is reported to be importantin denaturation of proteins in compressed CO₂; H₂O₂ itself is an oxidantand was used in a supercritical sterilization process; aqueous 1% tannicacid is an effective protein denaturant, widely used in allergyprevention; LS-54 is a carbohydrate surfactant with proven solubilityand micelle forming ability in scCO₂. Due to the low solubility ofwater, H₂O₂ and tannic acid in scCO₂, water-in-CO₂ micro-emulsion formedby LS-54 is an effective vehicle to deliver protein denaturant in thesubstrates. The inactivation results of these additives are shown inFIG. 2 (in order from left to right, the bars represent treatmentcompositions of: (1) Dry heat at 60° C.; (2) CO₂ and LS-54 at 60° C.;(3) CO₂, LS-54, and water at 60° C.; (4) CO₂ and water at 60° C.; (5)CO₂ and tannic acid at 60° C.; (6) CO₂ and hydrogen peroxide at 60° C.;(7) CO₂, LS-54, and tannic acid at 60° C.; and (8) CO₂, LS-54, andhydrogen peroxide at 60° C.).

Approximately 90% reduction of the enzymatic activity of trypsin wasachieved under two conditions, namely scCO₂+LS54+tannic acid (93.6%) andscCO₂+LS54+H₂O₂ (87.0%). Visual inspection of the processed glass vialreveal that the inner surface was completely wetted, indicatingsuccessful formation of microemulsion and penetration of microemulsionthrough the filters in the vial cap. These results proved the importanceof the surfactant in this trypsin denaturation process.

Dry heat at 60° C. did not reduce the activity of trypsin, indicatingthat trypsin is stable at temperatures up to 60° C. Trypsin processedwith scCO₂ and of LS-54 has the same activity as untreated trypsin.Deionized water+scCO₂, and the combination of LS-54+deionizedwater+scCO₂, have similar capacities to reduce trypsin activity tobetween 60% and 70%, confirming the observation of many otherresearchers that adding excessive amount of water causes partialdenaturation of proteins. This degree of inactivation has significantimpact on applications such as enzymatic catalyzed reactions. However,higher or complete inactivation is needed for the purpose ofinactivating unwanted proteins (allergens).

One percent tannic acid, a common protein denaturant, in, scCO₂ does notsignificantly reduce trypsin activity, compared to scCO₂+water andscCO₂+LS-54+water. This is an indication that tannic acid does not havesufficient solubility in scCO₂ to inactivate trypsin, so the majority ofthe inactivation observed with 1% tannic acid was due to the dissolvedwater in scCO₂. Adding 30% H₂O₂ into scCO₂ further reduced the proteinactivity to approximately 40%. This demonstrates that at least part ofthe protein was denatured from the oxidation reaction by the dissolvedH₂O₂ in scCO₂. Again, due to the low solubility of H₂O₂ in scCO₂, theH₂O₂ concentration is not sufficiently high enough to completelyinactivate trypsin.

Another series of preliminary experiments of CO₂+LS54+tannic acid wascarried out between 0° C. to 60° C. to explore the effects of processtemperature, because lower temperature is preferred to reduce energyconsumption and preserve the substrates (Error! Reference source notfound.). No significant inactivation of trypsin was observed attemperatures ≦40° C. Considering that trypsin loses most of its activityat 60° C., there seems to be a temperature barrier between 40° C. and60° C. in the current experimental settings indicating that there is asynergistic effect of CO₂ and elevated temperature.

One more preliminary experiment with scCO₂+LS54+tannic acid wasconducted with shorter exposure time (4 hours). Trypsin lost only 55% ofits activity, compared to the >90% of activity lost after 5 hours whichdemonstrates that long exposure is required to form LS54 microemulsionin scCO₂. Addition of agitation increases mass transfer from the liquidphase to supercritical CO₂, and might accelerate the formation ofmicroemulsion and reduce the time to inactivate trypsin.

In conclusion, the feasibility of reducing >90% activity of a modelprotein trypsin, with compressed CO₂ and selected surfactant and proteininactivation agents. This process is temperature dependent with asynergistic effect of CO₂ and elevated temperature. It is furtherillustrated that without agitation, long exposure is needed to achievehigh degree inactivation of proteins.

An exemplary allergen and endotoxin solubilization and removal apparatusis depicted in FIG. 4 . Gas for preparation of a high-pressure orsupercritical fluid is supplied to the CO₂ cleaning vessel 9 from gastank 1 via gas line 2. Gas line 2 can be an air-tight hollow tubeconstructed so as to withstand the required pressures for high-pressureand supercritical fluids. Pump inlet valve 3 can be positioned in fluidcommunication with gas line 2 and positioned intermediate gas tank 1 andCO₂ pump 4. Accordingly, pump inlet valve 2 can control the flow of gasfrom gas tank 1.

CO₂ pump 4 functions to provide gas from gas tank 1 to CO₂ cleaningvessel 9 as well as to provide the pressures for high-pressure orsupercritical fluids. In one aspect, CO₂ pump 4 can be calibrated toprovide a selected pressure. The CO₂ pump 4 is chilled by a watercirculator 5 to maintain a low temperature (e.g. 0° C.) to liquefy CO₂,in order to provide a high pumping efficiency. Feed valve 8 can controlthe flow of gas from pump 4 and is located along gas line 2 between thesecond water circulator 6 and CO₂ cleaning vessel 9. The second watercirculator 6 raises the CO₂ temperature to the desired cleaningtemperature. The second water circulator 6 also provide temperaturecontrol for the cleaning vessel through the heat-exchange coil 7 insidethe cleaning vessel.

The cleaning vessel is equipped with a internal impeller 10, whichprovides agitation to the CO₂ phase and the substrates to be cleaned.Liquid additives 11 are added to the bottom of the cleaning vessel 10.The additives 11 will dissolve into the CO₂ phase and formmicroemulstions which solubilize allergens and endotoxin and remove themfrom the cleaning substrates. The pressure inside the cleaning vessel ismonitored by a pressure gauge 12. Safety head 13 is connected to preventover pressurization of the cleaning vessel 9. Vent valve 14 is used torelease the CO₂ pressure inside the cleaning vessel at the end of acleaning operation.

Example of Endotoxin Solubilization and Removal

Materials:

E. coli 055:B5 endotoxin and limulus amebocyte lysate (LAL) kinetic-QCLassay kit were purchased from Lonza Walkersville Inc. (Walkersville,Md.). HyPure™ cell culture grade water (endotoxin-free water) waspurchased from HyClone Laboratories Inc. (Logan, Utah). Dehypon Ls-54surfactant was a gift from Cognis Corporation (Ambler, Pa.).

Quantifying Endotoxin

The level of endotoxin was quantified with a standard LAL assay.Endotoxin catalyze the conversion of a proenzyme in LAL to its enzymaticform. This enzyme is capable of decompose a colorless substrateAc-LLE-Glu-Ala-Arg-pNA to p-nitroaniline (pNA) which absorbs light at405 nm. The rate of the color change cause by the production of pNA isused to quantify the endotoxin level.

Procedure

Titanium discs (12 mm diameter, 2.5 mm thickness) were used in theexample to demonstrate the effectiveness of the disclosed endotoxincleaning process. Each disc was coated with approximately 2000 unites ofendotoxin (˜0.2 microgram of endotoxin). Four discs (three coated, oneclean) were glued to the impeller, facing downward. Twelve grams ofwater and 32 grams of LS-54 surfactant were added to the bottom of thecleaning vessel. After the cleaning vessel was closed, a CO₂ pumpstarted to pressurize the vessel with CO₂ until reaching thepredetermined pressure (2000 or 4000 psi in this example). Thetemperature was held at constant temperature (5° C. or 40° C. in thisexample).The impeller was turned on (400 rpm) to provide agitation, inorder to facilitate the formation of water-in-CO₂ microemulsions and thesolubilization of endotoxin into the microemulsion.

The temperature, pressure, and agitation were maintained for thepredetermined amount of time. Then the feed valve was closed. Thecleaning vessel was slowly depressurized through the vent valve untilthe vessel reached atmospheric pressure. The impeller was immediatelyremoved from the cleaning vessel. All attached discs were retrieved todetermine the remaining endotoxin level with LAL assay.

Results and Discussion

Various combinations of temperature, pressure and processing time havebeen tested, as summarized in Table 1:

TABLE 1 Time Initial loading Cleaning Fluid (s) T (C.) P (psi) (hr)(EU/disk) Supercritical (SC) CO₂ 40 4000 4 2628 scCO₂ + Ls-54 & water 404000 4 2628 liquid CO₂ 5 4000 4 2428 ± 71  liquid CO₂ + Ls-54 & water 54000 4 2370 ± 82  liquid CO₂ + Ls-54 5 4000 4 3023 ± 457 liquid CO₂ +water 5 4000 4 2627 ± 265 liquid CO₂ + Ls-54 & water 5 2000 4 2169 ± 810liquid CO₂ + Ls-54 & water 5 4000 2 3145 ± 438

Error! Reference source not found. shows the percentage of endotoxin atdifferent testing conditions. High levels of endotoxin removal have beenachieved by adding Ls-54 and water to both supercritical (SC) CO₂ (81%)and liquid CO₂ (93%). Liquid CO₂+Ls-54 and water removed a greaterfraction of endotoxin than sc CO₂+Ls 54 and water, because Ls-54 hashigher solubility in compressed CO₂ at lower temperatures and constantpressures.

The results indicate that LS-54 microemulsions were formed in CO₂ phaseand the water in the microemulsions was able to remove significantamount (up to 93%) of endotoxin. (See FIGS. 5 and 6). The higherefficiency achieved in the liquid state (ρ=23.390 mole/L) indicates thatmore microemulsions are formed in this state than in the supercritical(ρ=20.376 mole/L). Neither seCO₂ nor liquid CO₂ removed a significantfraction of endotoxin from the Ti surfaces, because CO₂ does notdissolve endotoxin.

It was also desired to verify the impact of liquid CO₂+Ls-54 and waterin removing endotoxin from the metal surfaces decreasing either the timefor treatment or the pressure set before (4 hr and 4,000 psi). Error!Reference source not found. shows the results for this set ofexperiments in terms of percent removal. The average endotoxin levelafter 2 hour treatment and 4,000 psi was 1296+189 EU/disk with a percentremoval of 59±2.4%. For 4 hour treatment and 2,000 psi the averageendotoxin remaining was 1004±653 EU/disk with an average percent removalof 57±8%.

Both treatments achieved the same efficacy. However, none of themachieved the efficacy obtained for the 4 hour treatment and 4000 psi(93%). It is noticeable that decreasing either the time or the pressurefor treatment will decrease the endotoxin removal in this system.However, it needs to be pointed out that for this system a well-mixedsolution for the cleaning fluids is not expected since stirring is notprovided. The shaft holding the disks is allowed to spin within thesolution, but complete mixing of the fluids and agitation forces is notguaranteed.

These and other modifications and variations to the present inventionmay be practiced by those of ordinary skill in the art, withoutdeparting from the spirit and scope of the present invention, which ismore particularly set forth in the appended claims. In addition, itshould be understood the aspects of the various embodiments may beinterchanged both in whole or in part. Furthermore, those of ordinaryskill in the art will appreciate that the foregoing description is byway of example only, and is not intended to limit the invention sofurther described in the appended claims.

What is claimed:
 1. A method of making a treatment composition, themethod comprising: loading a substrate into a high pressure vessel;loading active components into the high pressure vessel, wherein theactive components comprise a protein denaturant and a surfactant; andthereafter, pressurizing the high pressure vessel with carbon dioxide toreach a pressure within the high pressure vessel of about 400 pounds persquare inch to about 1,070 pounds per square inch.
 2. The method as inclaim 1, wherein the active components further comprise an acaricide. 3.The method as in claim 2, wherein the acaricide comprises permethrin,benzyl benzoate, or a mixture thereof.
 4. The method as in claim 1,wherein the protein denaturant comprises ethanol, hydrogen peroxide,benzyl benzoate, tannic acid, or a mixture thereof.
 5. The method as inclaim 1, wherein the surfactant comprises a fluorosurfactant, an ionicsurfactant, a silicon-based surfactant, a hydrocarbon surfactant, or amixture thereof.
 6. The method as in claim 1, wherein the carbon dioxidehas a purity of about 90% by volume or more.
 7. The method as in claim1, wherein the carbon dioxide loaded into the high pressure vessel issubstantially pure carbon dioxide gas.